G protein-coupled receptors are ubiquitous components of signal transduction pathways, including many neurotransmitter systems. This project is designed to assess the role of polyunsaturated phospholipids in modulating G protein-coupled signal transduction and to elucidate the mechanism of action of ethanol in these systems. The visual transduction pathway of the retinal rod photoreceptor is being used as a model system. System properties we are studying include: 1. the kinetics and extent of formation of metarhodopsin II (MII), the G protein activating form of rhodopsin; 2. MII/G protein complex formation; 3. the rate of G protein activation; 4. cGMP phosphodiesterase (PDE) activation; and 5. the GTPase activity of the G protein. Both functional measures in the transduction pathway and acyl chain packing properties of the phospholipid bilayer are being investigated. Current studies demonstrate that the kinetics and extent of formation of the MII-G protein complex are dependent on both acyl chain formation and cholesterol content. In particular the kinetic coupling of MII to G protein is slowed by 50% in 18:0,18:1PC relative to 18:0,22:6PC. The addition of cholesterol doubles the lag time in complex formation in 18:0,18:1PC, whereas the lag time is essentially unchanged upon the addition of cholesterol to 18:0,22:6PC. In other experiments, it is shown that the PDE activity, a measure of the integrated visual pathway function, is also dependent upon acyl chain composition. Here again, 22:6n-3 phospholipids yield the highest levels of activity. The role of lateral domain formation in signaling systems was investigated using acyl chain specific fluorescence probes and fluorescence energy transfer techniques. These studies demonstrate that rhodopsin prefers an environment enriched in polyunsaturated acyl chain lipids relative to a more saturated phospholipid environment. Taken together, these studies indicate that 22:6n-3 containing phospholipids enhance the efficiency of a G protein-coupled signaling system and appear to impart unique structural properties to bilayers in the form of lateral domains. These studies provide a basis for understanding the visual and cognitive deficits associated with a nutritional deficiency of n-3 fatty acids.